Lung cancer is the most lethal of human tumors due to both its aggressive biological behavior and the lack of early detection. Improved survival depends on targeting high risk individuals coupled with advancements in tumor detection methods. Our recent findings identify three research areas with significant potential for biomarker development and which would positively impact detection of high risk individuals and/or invasive tumors. Our hypotheses are multifaceted. First, chromosomal instability is a fundamental feature of malignant transformation in the lower airway and is an abnormality detectable by FISH analysis of chromosome copy number. Second, RNA splicing is highly variable in lung tumors for proteins such as Muc-1 and these splicing abnormalities may be a consistent feature of epithelial malignancies. Detection of Muc-1 splicing variants using RT-PCR or ELISA assay for translated splice variant peptide represents a new and innovative approach to tumor cell detection. Third, numerous gene expression abnormalities occur in lower airway epithelium during lung carcinogenesis. We have identified several differentially expressed genes in lung tumors including WNT5a, and HOXA9 and have isolated other genes (SEMA3F and hDEF- 3) implicated by genetic deletions. Global evaluation of gene expression patterns should identify many more candidates for biomarker development. Our specific aims therefore will: 1) assess chromosome copy number and/or region specific instability in malignant and premalignant airway epithelial cells using spectral imaging technology, 2) confirm the specificity of alternate Muc-1 RNA splicing in lung tumors and develop antibody reagents for improved detection, 3) evaluate differentially expressed WNT and HOX loci, along with SEMA3F, hDEF-3 and HuD proteins for use as biomarkers and 4) identify more differentially expressed genes using cDNA arrays or powerful new differential display approaches. These last techniques will be applied to lung specimens derived from tumors, biopsies or primary cultures of normal and preneoplastic epithelium and possibly abnormal cells derived from sputum or blood. Patterns of gene expression likely represent the most detailed molecular characterization of tumors and preneoplastic cells currently possible and should provide a host of new biomarkers for evaluation. Initial expression profiles of HOX, WNT and FGF gene families have identified interesting differences between normal lung and tumor cells. Selected genes of interest will be developed further by generation of immunological reagents for protein detection. All testing results will be pooled into a common clinical database for correlation, where appropriate, with standard cytological and histopathological results and with clinical outcomes. A specimen processing facility for the preparation of cells from sputum and blood will distribute specimens to testing laboratories. This facility will employ new flow cytometry, centrifugation and immunomagnetic fractionation procedures for bronchial washings and lavage fluid, sputum and peripheral blood.